Laser cell purification system

ABSTRACT

A process and apparatus for cell purification and ablation is disclosed. The present invention comprises a laser system which directs radiant energy at computer or manually selected individual cells thereby disrupting DNA, RNA and protein structure in those cells. The present invention produces a purified tissue section containing relatively intact DNA, RNA or protein from only the untreated cells. This purified sample is suitable for amplification of material by PCR or other techniques for the analysis of molecular genetic features in the selected cells of interest.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of Ser. No. 08/530,791, filed Sep. 19,1995, now U.S. Pat. No. 6,040,139.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention involves a novel method and apparatus for obtaining purecell populations or cell constituents such as DNA, RNA or proteins fromtarget cells in tissue sections using ultraviolet (UV) laser-assistedablation of non-target cells.

2. Description of the Background Art

Cancer is a leading cause of death in the United States. Treatments forcancers include surgery, chemotherapy, and radiation therapy, whichcause considerable morbidity and often are ineffective. Standardpathological grading and staging cannot predict the susceptibility of aparticular tumor to eradication by chemotherapy, radiation therapy, orother therapy, and thus many patients with solid tumors receiveineffective toxic therapy. Better prognostic indicators and therapeutictargets are needed for cancer treatment.

A large worldwide effort is underway to develop improved prognostic andtherapeutic tools for cancer. New molecular biology techniques permitinvestigation of specific genetic alterations in cancers. Evidence isaccumulating that information about specific DNA alterations in tumors,which predict cancer behavior, may provide important new tools forcancer diagnosis, prognosis, and therapy.

For example, a recent study found that in one of the most common cancersin children (Wilms' tumor), tumor-specific loss of heterozygosity ofchromosome 16q predicts adverse outcome independent of histologicaltype. Based on this knowledge, the subgroup of Wilms' tumor patientswith favorable histology and loss of heterozygosity for chromosome 16qin their tumors may now benefit from earlier, more aggressive therapy.Further, in colon cancer, the status of chromosome 18q has recently beenshown to have strong prognostic value in patients with cancer extendingthrough the bowel without lymph node metastasis (stage II). Thus, stageII colon cancer patients with tumor specific loss of heterozygosity onchromosome 18q are a newly defined subset of patients that may benefitfrom more aggressive adjuvant therapy at the time of their initialdiagnosis.

Other types of tumor-specific genetic alterations, includingamplification of specific alleles and inactivation of specific genes oralleles by cytidine methylation have shown promise for providingimportant new prognostic information. It may be possible to define the“signature” of genetic lesions in an individual patient's tumor,permitting therapy tailored specifically to the genetic defects of thetumor.

Current molecular biologic techniques allow the study of DNA, RNA, andprotein contained within cells. Some techniques allow cells to bestudied in situ, with labelled molecular probes visualized under amicroscope. These techniques are very useful, but currently are quitelimited in their resolution and consistency. Other more powerfultechniques for studying cellular DNA, RNA or protein depend on poolingof material from one or several cells. Studies based on such poolinghave identified the first known gene-specific changes associated withcancer and other diseases, and have provided insight into the molecularprocesses involved in the transformation of cells from normal toabnormal.

Understanding molecular genetic changes involved in the pathogenesis oforgan dysfunction requires studying groups of diseased cells inisolation and comparing them to phenotypically normal cells. Thedifficulty is that diseased cells in any tissue are usually accompaniedby many phenotypically normal cells. Thus molecular studies reported todate have been limited to tissues in which the concentration of diseasedcells is relatively high, such as in large, concentrated tumor masses.Various researchers have attempted to obtain purer samples of DNA, RNA,or protein from diseased cells by scraping portions of tissue sectionsaway with a cutting instrument or by inking target areas of tissuesections and later exposing the section to UV light to destroy non-inkedDNA and RNA.

Ultraviolet irradiation of such tissue has been found to cause singleand double stranded DNA breaks, DNA crosslinks, generation of localdenatured sites in DNA and DNA base destruction. Thus, it is known thatultraviolet irradiation of a tissue section can massively disrupt theDNA strand (as well as RNA and protein) contained within that tissuesection. For example, selective UV irradiation (non-laser) exposure ofportions of tissue sections was achieved by Shibata et al. (Amer. J.Pathol. 141(3):539-543, 1992) by covering target areas of stained tissuesections with black ink and UV irradiating the entire tissue sample witha standard broad spectrum UV light bulb. Shibata demonstrated that DNAwithin cells covered with the black ink is preserved, while DNA in UVexposed adjacent portions of the tissue was destroyed. This crudetechnique is markedly limited by the width of the black marking penused, by difficulty in directing the pen to the desired location, and bythe need to continually replace the pen in order to avoid contaminationof inked areas by cellular material from areas previously inked. Anotherlimitation is that inking must be performed while no cover slip is inplace, markedly reducing optical resolution and making visualidentification of cells nearly impossible in many cases.

Formalin fixed, paraffin embedded (FFPE) tissues are the basis forcurrent pathology practice. They are readily available to mostpathologists and cancer researchers and provide histological detail thatremains the benchmark for pathology. FFPE tissue is not ideal for manymolecular methods because DNA and RNA contained within this tissue ispartially degraded. Although it is more difficult to isolate DNA ofadequate quality for analysis from FFPE tissue sections than fromunfixed, unembedded tissue, a number of studies have demonstrated thatamplification of DNA fragments as long as 536 base pairs can beaccomplished with tissue fixed in buffered formalin. However, theduration of storage, fixative used, fixation time, fixation temperature,and extraction procedures all affect the quality of DNA that can beisolated from paraffin. Recent molecular techniques have allowed a widerange of genetic alterations to be detected in DNA and RNA isolated fromarchival tissues. Most of these techniques are based on Polymerase ChainReaction (“PCR”).

PCR based genetic analysis of single cells or groups of cells has beenused to discover molecular alterations in cells. For example, PCRtechniques have been used to detect loss of heterozygosity, genomic DNAmutation, mitochondrial DNA mutation, DNA methylation, gene dosage, generearrangements, clonality and detection of DNA adducts. However, becausecancer cells grow in close relation to noncancerous cells in alltissues, it is nearly impossible using heretofore known techniques toobtain pure tumor DNA. Hence, background signals from noncancerous cellsoften distort the analysis of genetic changes in tumors. For example,when a mutation is not detected in a particular gene in DNA isolatedfrom a tumor, it is quite possible that the nonmutated sequence camefrom noncancerous cells' DNA contaminating the sample. Thiscontamination problem was demonstrated in a controversy concerning theimportance of the recently identified gene p16/MTS1. One of the gene'sdiscoverers cast doubt on analyses of certain DNA samples which did notshow p16 mutations because of contamination by noncancerous DNA.

The problem of background noise created by contaminating noncancerouscells was again emphasized by difficulty in identifying mutations in thebreast cancer associated gene BRCA1 in sporadic and hereditary tumors.In cases of hereditary tumors, the individual inherits one mutated copyof the gene. Researchers have had difficulty studying the remaining copyof the gene in hereditary tumor samples because of background noise fromcontaminating normal cells, making it difficult to ascertain thefrequency of specific BRCA1genetic alterations. Because of thedifficulty of obtaining pure breast cancer DNA samples in general, it isnot known with confidence that in fact BRCA1mutations are rare innon-hereditary breast tumors. Interpretation of results based on impuretissue samples are ambiguous in direct proportion to the degree ofimpurity of the DNA, RNA or protein isolated.

Current techniques of DNA, RNA and protein isolation and purificationillustrate the problem of sample purity in researching geneticalterations in cancer.

For example, serial cryostat sectioning and trimming of frozentumor-bearing tissue has been used to produce limited purification oftumor DNA and has been useful in the definition of loss ofheterozygosity and other genetic alterations. Unfortunately, thiscryostat-based “fractionation” of cancer tissue has a number ofdrawbacks. For example, most common primary tumors, including breast,ovarian, pancreatic, prostate cancers and others, are highly infiltratedwith noncancerous cells. Hence, it is difficult to obtain better than70% purity in the majority of cases using cryostat sectioning alone,since only relatively large regions of the tissue can be carved manuallyfrom the tissue block. Further, histological detail is often poor infrozen sectioned material, making interpretation difficult. In addition,cryostat methods cannot be applied easily to the clinical settingbecause clinicians find it inconvenient to freeze biopsy samples, andpathologists prefer paraffin embedded material for interpretation.

Improved signal (cancerous DNA) to noise (noncancerous DNA) ratio in PCRbased allelic loss analysis has been achieved by mechanicalmicrodissection of individual tissue sections or by a combination ofbroadband ultraviolet light treatment of the section after coveringdesired areas with black ink, followed by mechanical removal of thetissue from the slide. However, these manual microdissection methodshave several drawbacks. For example, microdissection or ink-dotting mustbe performed without a cover slip in place which markedly reducesoptical resolution. Further, manual dissection or ink dotting onlyallows the selective isolation of clumps of cells.

Microdissection using stage-mounted micromanipulators has been reported,but the lack of resolution without a coverslip in place typicallyprecludes isolation of single cells. Moreover, the microdissection andink-dotting techniques are extremely time-consuming and cumbersome.

In situ hybridization is another method of locating specific basesequences in tissue sections. For example, fluorescence in situhybridization (FISH) has been used to show sequence deletions and ploidyanomalies in prostate tumors. However, in situ methods have severaldisadvantages. For example, genetic material in the section is consumedby the process and is not available for multiple analyses. In addition,the specificity of individual FISH probes is highly variable. Finally,many molecular alterations (mutations, methylation, small deletions,etc.) are not detectable with FISH.

Non-histological methods for enrichment of tissue samples of tumor cellsare also possible, such as separation of ovarian tumor cells using flowcytometry, and enrichment of breast and prostate tumor cells with anavidin affinity column. However, these techniques are significantlylimited because they require a tumor specific antibody, which is notavailable for the majority of tumors. Moreover, even if tumor specificantibodies were available for all tumors, use in a clinical settingwould require separate separation protocols and columns for every tumortype, which would be very inefficient. Furthermore, specific antibodiesare notoriously variable in their staining characteristics. Finally,these “blind” separation methods make it difficult to obtain bothhistological grading and molecular diagnostic information on the sametissue sample. Histological grading of tumors is one of the mostpowerful prognostic tools available and any new molecular analysis willbe compared to standard histological grading techniques to determinetheir prognostic value, a comparison that will not easily be achievedwith flow cytometry based or affinity column based purification systems.A method that combines the ability to obtain maximum enrichment of tumorcells while maintaining histological analysis of the sample hassignificant advantages over flow cytometry or column-based separationmethods.

U.S. Pat. No. 5,272,081 discloses a method for selecting and separatingindividual cells in a sample for diagnostic purposes. In accordance withthis method, cells are first separated, trapped in sized holes on agrid, each with a known location, and subjected to tests for selection.Once the cells on the grid have been tested and selected, the desiredcells may be removed from the grid by selectively changing theelectrical potential of conductors on the grid, or the undesired cellsmay be killed to effect separation. A major disadvantage of this methodis that the cells must be suspended before they are trapped on the gridfor testing. Cells in a standard paraffin embedded tissue sample, suchas a biopsy sample, cannot be readily kept intact and separated by thismethod. Again, standard histological analysis of the same sample wouldbe impossible, and usually the entire sample would be consumed by oneexperiment.

Also, U.S. Pat. Nos. 4,624,915 and 4,626,687 disclose methods anddevices for the separation and segregation of living cells using afocused radiant energy beam. In accordance with these methods, living,anchorage-dependent target cells are moved on a microscope stage in twodimensions while the laser beam is directed through the objective lensof the microscope at individual living cells, or at the film supportingthe cells. The living cells are viewed on the slides with no cover slipand while nutritive cell medium is flowing over the cells. Selection isbased on a cellular light response to an attenuated laser beam, andundesired cells are killed with radiant energy from a focused, highpower laser beam. Living cell selection can be accomplished by computeranalysis of the fluorescence pattern of the living cells.

Freeze-dried tissue sections have been microdissected using a laserafter first storing coordinates to be dissected from a non-freeze driedadjacent section using a drawing tube (Klimek et al., Carcinogenesis:5(2):265-268, 1984). Because the tissue sections to be microdissectedmust be freeze-dried at −40° C. and stored under vacuum at −45° C.,because the tissue shrinks when it is freeze dried, and because theregions to be dissected must be indirectly identified through the use ofa drawing tube, this method is also very cumbersome and limited inprecision. It does not allow specific individual cellular localizationand ablation.

None of the known techniques contemplated for use with biopsy tissuesamples have been used to reliably examine and selectively destroyindividual cells, individual cell nuclei, or DNA within individualcells. The known techniques make it nearly impossible to routinely studysmall tissue samples in which there are a large percentage ofnon-diseased cells that cannot be cut out or selectively inked. Prostatebiopsies, for example, may contain small foci of adenocarcinomameasuring 1 mm in diameter or less, surrounded by lymphocytes, smoothmuscle and benign prostate cells. In most cases, it is extremely tediousor impossible to selectively destroy the lymphocytes, smooth musclecells, and benign prostate cells using available techniques. Theselimitations make it difficult to bring routine pathologic diagnosis tothe molecular level, because most tissue biopsies cannot be effectivelystudied using currently available techniques.

Using current tumor DNA purification techniques, noise fromcontaminating noncancerous DNA makes interpretation of data difficultand sometimes impossible, confounding cancer research and diagnosis.Accordingly, there is a profound need for a reliable technique to obtainhighly enriched samples of tumor DNA, RNA and protein. Such a techniquewould be widely used by pathologists in clinical practice, cancerresearchers, and in fact, any investigator who needed to purifyindividual cells or groups of cells from a heterogeneous tissue,cancerous or noncancerous.

SUMMARY OF THE INVENTION

In accordance with the present invention, a novel laser purificationmethod and apparatus is disclosed for obtaining pure cell populationsfrom a tissue section having a heterogenous cell population. This methodallows one to obtain a tissue section having a heterogenous cellpopulation which, after treatment, has relatively intact DNA, RNA orprotein from only untreated cells. The invention comprises a microscope,a support stage for supporting the tissue section and a drive means forproviding relative movement between the stage and the microscope in anx-y coordinate plane. A video camera is provided for scanning the tissuesection and creating a mosaic of video images representing the tissuesection. These high resolution video images are displayed on a videodisplay. Target cells in the tissue section are microscopicallyidentified using established morphological criteria or fluorescenttagged antibodies. In one embodiment, the operator inspects the videoimages representing the magnified tissue section displayed on the videodisplay and manually selects the target cells to be destroyed orpreserved utilizing a touch or light sensitive video display screen orcursor system. In another embodiment, the target cells of interest areautomatically identified based upon an analysis of the video images by acomputer. In this embodiment, the operator can verify the target cellsidentified by the computer and has the option of either selectingadditional cells of interest or deselecting cells identified by thecomputer. After the manual or automatic identification and selection ofthe target cells, the computer automatically directs a laser tophotoablate the DNA, RNA or protein in the contaminating cells so thatthe proportion of intact DNA and RNA from the target cells is markedlyincreased.

Further purification of the tissue section is achieved with the presentinvention by analyzing the cells not photoablated in the purificationsteps described above under a higher magnification, and repeating thephotoablation process of the present invention. Purification, inaccordance with one aspect of the present invention, means that thetissue section having a heterogenous cell population, after treatment,will contain relatively intact DNA, RNA or protein from only theuntreated cells.

The present invention is automated by using image analysis hardware andsoftware, with minimal operator intervention, thereby allowing the DNAor RNA in selected portions of the tissue section to be automaticallyablated, while the nonselected cells' DNA or RNA will remain intact forsubsequent analysis by PCR or other genetic methods.

Further, a greater degree of optical resolution is obtainable with thepresent invention over prior art systems since a UV transparent quartzcover slip can be used during operation of the viewing and laserdevices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the laser purification system inaccordance with the present invention.

FIG. 2 is a schematic representation of the objective, quartz coverglassand tissue support of the laser purification system of FIG. 1.

FIG. 3 is a schematic representation of the video display and theapparatus for manually selecting target cells from the tissue section inaccordance with the present invention.

FIG. 4 is a block diagram illustrating the laser purification system inaccordance with the present invention.

FIG. 5 is a flow chart illustrating the operation of the laserpurification system in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The laser purification system of the present invention is described inconnection with FIGS. 1 and 4 which disclose a microscope 10 for viewinga tissue section 12 which contains a heterogenous population of cells,including target cells of interest, such as cancerous cells. Themicroscope 10 comprises an objective lens 18. The tissue section 12 issupported on a tissue support 14 which can be any suitable means forsupporting a tissue section known to those skilled in the art, such as aslide. In one embodiment, the tissue support is a borosilicate glassslide.

In a preferred embodiment, a UV transparent quartz coverslip 15, asillustrated in FIG. 2, is placed between the tissue section 12 and theobjective 18 which allows a greater degree of optical resolution. In oneembodiment, the quartz coverslip 15 is approximately 0.15 mm to 0.25 mmthick or even thicker.

A suitable UV transmitting mounting medium 13 is placed between thecoverslip 15 and the tissue section 12 and tissue support 14. Anymounting medium that allows at least partial UV light transmission, isclear (to allow good visualization), and does not interfere with laterDNA, RNA, or protein recovery or analysis is suitable for use with thisdevice. For example, one acceptable UV transparent mounting medium issterile distilled water.

The tissue section 12 and tissue support 14 are removably mounted on astage 16. The stage 16 comprises a drive means for providing relativemovement between the stage 16 and the objective lens 18 in incrementallocations in an x-y coordinate plane. The drive means also comprisesmeans for generating an address signal within the x-y coordinate planefor each incremental location.

The drive means in accordance with the present invention can be anymotorized stage known to those skilled in the art which providescontrolled incremental movement of the stage and generates an addresssignal representing the incremental location. As illustrated in FIG. 4,the drive means includes stage controls 21 to control the movement ofthe stage 16 in response to address signals. In a preferred embodiment,the drive means is a motorized stage with computer interface. Amotorized stage suitable for use with the present invention can beobtained by Ludl, Inc. such as, for example, a motorized stage having aresolution of approximately 0.1 um, repeatability of approximately 1 um,and accuracy of approximately 5 um. Of course, other motorized stageshaving greater or lesser accuracy, repeatability and resolution may beused as well.

The microscope 10 can be any suitable microscope known to those skilledin the art, such as an Olympus BX40 upright microscope. The Olympus BX40microscope is an economical instrument with the key advantages ofmodular construction and infinity corrected optics, which eases theintegration of the laser 22 to the microscope 10. Special UV transparentquartz objective lenses, such as those illustrated by objective 18, maybe acquired from Optics for Research, Inc. or other suitable source.

As the tissue section is incrementally moved by drive means and stage16, video camera 20 generates a video image of the tissue section atselected incremental locations in the x-y plane. In a preferredembodiment, the video camera 20 is mounted to microscope 10 asillustrated in FIG. 1. The signals generated by the video camera 20representing each video image are input into a video frame grabber 35which are then input into the computer 30, as illustrated in FIG. 4. Theaddress signal of the stage 16 associated with each video frame areinput into computer 30.

Image focus can be maintained manually by the operator by mechanicallyfocussing the microscope 10. In a preferred embodiment, the image focusis maintained by automatic image focussing equipment which is well knownto persons skilled in the art.

The video camera 20 can be any suitable color or monochrome video cameraknown to persons skilled in the art and is preferably a Sony XC-75monochrome instrumentation camera or a high resolution color camera. Inone embodiment of the present invention, the video camera 20 feeds up to30 frames per second to the video frame grabber 35. The video framegrabber 35 can be any suitable video frame grabber known to personsskilled in the art and, in a preferred embodiment, is a Data TranslationQuickcapture video frame grabber. Further, the computer 30 can be anysuitable computer known to persons skilled in the art and, in apreferred embodiment, is a Apple PowerMac 8100 which has a 16 MB RAM anda 500 MB hard drive. A Pentium PC may also be used.

The present invention also comprises a selection means for selecting thetarget cells of interest from the heterogenous target tissue section 12.Selection is defined as choosing specific regions defined by specificx,y coordinates. In one embodiment, the selection means automaticallyselects the target cells of interest to be photoablated by a focussedradiant energy beam 23, as illustrated in FIG. 1. In accordance withthis embodiment, the selection means comprises the computer 30 and avideo display 33 which utilizes a light or touch sensitive screen, asillustrated in FIGS. 3 and 4. The computer 30 is programmed to analyzethe video images from the video camera 20 and to identify target cellsof interest based on various histological or biological characteristicsor fluorescent characteristics of those cells selected by the operator.For example, the target cells could be identified using establishedmorphological criteria or fluorescent tagged antibodies, as describedbelow.

In another embodiment of the present invention, the selection meanscomprises a means for the manual selection of target cells of interest.In accordance with this embodiment, the computer 30 displays the videoimages generated by video camera 20 on a video display 33 (FIG. 3),creating a mosaic of the video images representing at least a portion ofthe entire tissue section. The operator then inspects the highresolution video images of the tissue section and manually selectstarget cells to be treated or target cells to be preserved. The manualselection of the target cells is accomplished in this embodiment byutilizing a touch or light sensitive screen in video monitor 33 or byusing a cursor system or mouse. For example, the operator identifies thetarget cells of interest by tracing those cells using a light pen 34.

FIG. 3 illustrates a diagram representing a mosaic of high resolutionvideo images representing a magnified tissue section containing aheterogenous population of cells, including non-cancerous cells andadenocarcinoma cells (gland forming cancer). The ring of gland formingcancer cells in this tissue section is represented by the numeral 37 andnon-cancerous ring of prostate gland cells is represented by numeral 39.Further, noncancerous stromal cells are depicted as numeral 40 andnon-cancerous lymphocytes are depicted as numeral 41. In this example,the operator can use the light pen 34 to outline the target cells ofinterest to be ablated, such as non-cancerous cells 39, 40 and 41. Inaccordance with the present invention, the operator can also elect tooutline cells which are not to be ablated, such as by outlining thecancerous gland forming cells 37 in FIG. 3.

The computer identifies the specific address for each target cell ofinterest identified either automatically or manually by the operator.

After identification and selection of the target cells of interest, thepresent invention comprises a radiant energy beam generating means forautomatically and selectively ablating only those target cellsidentified automatically or manually by the operator. Specifically, theradiant energy beam generating means automatically directs a radiantenergy beam to photoablate the DNA, RNA or protein in the unwanted,contaminating cells. In a preferred embodiment, the radiant energy beamgenerating means generates an ultraviolet (“UV”) laser beam 23 by UVlaser 22.

The UV laser 22 can be any suitable laser known to those skilled in theart such as, for example, argon ion lasers, excimer pumped dye lasers,Nd:YAG lasers, KrF excimer lasers and XeCl lasers. In one embodiment theUV laser is a diode pumped solid state Nd:YLF laser which produces acollimated beam with a Gaussian intensity profile. The beam diameter canbe any suitable beam diameter for causing photoablation of cells as isknown to those skilled in the art. For example, the beam diameter can beany suitable diameter from less than approximately 1 micron toapproximately 1 mm. In one embodiment, the beam diameter isapproximately 10 um, which is the average diameter of human cell nuclei.

UV light is specifically selected for its known tendency to damagecellular DNA, RNA and protein. Laser light of any wavelength within theUV spectrum will be suitable for proper functioning of the presentinvention. UV laser light in the spectrum designated UV-C (200-290 nm)has the greatest potential for DNA, RNA and protein damage with thelowest energy applied and least heat production. Laser light with awavelength above 290 nm could also be used, but may produce tissueboiling at lower energy levels.

The laser 22 can be controlled by the operator, through computer 30, toselect the specific pulse duration, wavelength and energy level of thelaser beam 23. In one embodiment, the laser 22 produces pulses having aduration of less than about 7 ns and no greater than 200 microJoules at262 nm. In another embodiment, the laser 22 produces pulses at 248 nm.When the laser 22 is focused through a microscope objective to a spot ofapproximately 0.5 um diameter, the maximum optical power (fluence)exceeds 100 kiloJoules/cm² per pulse. The maximum fluence available isvastly greater than required, and fluence can be decreased to any leveldesired by the user. Conservative estimates suggest that organicmaterials such as biological tissues are completely ablated with 0.02 to1 Joules/cm². Ceramics, glass and metals are ablated with 1 to 20Joules/cm²

The UV laser 22 is controlled by the computer 30, which issues commandsignals to initiate a burst of optical pulses 23. The laser output isbrought to the laser port through a series of mirrors 24 and lenses, asillustrated in FIG. 1. A beam expander 28, controlled by the computer30, is also provided so that laser beam spot size can be accurately andprecisely controlled. The collimated and expanded UV laser beam 23 fillsthe back aperture of the objective lens 18, thus creating adiffraction-limited spot at the tissue section plane.

A dichroic mirror 27 is used at the position between the video camera 20and the microscope objective lens 18, so that the tissue section can beviewed when the laser is active. The dichroic mirror 27 reflects lightat wavelengths less than 300 nm (in one embodiment), but transmitsvisible light, protecting the operator from stray UV light at theeyepieces. Additional laser safety features include a keylock controlledpower switch and a manual occluder at the laser output port. In oneembodiment, a UV blocking swing-out shield is placed around the stageand contains a switch that will prevent laser firing while the shield isopen.

The size of the laser spot at the tissue section depends on the diameterof the laser beam as it enters the optical train into the microscope 10.Beam expander 28 between the laser 22 and the microscope 10 allows thespot to be varied to increase or decrease the treatment area. The beamsize can be selected by the operator and the computer 30 effectuatescontrol of the beam size by controlling beam expander 28. The maximumtreatment area is the field of view of the microscope 10 which dependson the numerical aperture of the objective lens in place. The minimumspot size obtainable depends on focal length of the objective lens. Ifthe fluence used is sufficient only to produce inactivation of thetarget cells'RNA, DNA or protein in the target zone, the effect ofscattered light will be minimized. Optimal laser fluence and exposuretime will be determined by the user and will vary depending on theapplication (some tissues may requires higher UV doses to ablateDNA/RNA, for example).

The applied dose of UV radiation is controlled by the attenuator 29,varying the duration and number of pulses applied to the target zone,and varying the diameter of the spot. The operator can control theseparameters by inputting the desired information into computer 30 throughterminal 36 (FIG. 4) which controls the laser 22, beam expander 28 andbeam attenuator 29.

The present invention utilizes any suitable quartz laser-to-microscopeoptics. For example, suitable quartz laser-to-microscope optics can beobtained from Microcosm, Inc. (a Maryland corporation). In oneembodiment, the beam expander 28 and the laser 22 utilize serialinterface electronics and software extensions to NIH-Image software(which is publicly available) to enable beam size and laser pulse trainsto be computer controlled. In a preferred embodiment, the beam expander28 is of Galilean design with variable magnification to control spotsize. The magnification adjustment is motorized and controlled from thecomputer.

Operation of the Present Invention

The operation of the present invention will be described in connectionwith FIG. 5. An appropriately stained tissue section covered with a UVtransparent quartz coverslip 15 is placed under the histologicmicroscope 10 with attached high resolution camera 20. The tissuesection can be any suitable thickness known to persons skilled in theart such as, for example, a 6 micron thick section. The user selects theobjective to be used (10×, 20×, 32× or 100× for example) and initiatesthe scanning function of the device. The tissue section then is moved inan x-y plane underneath the microscope by stage 16 and the drive means.The video camera 20 generates a video frame of each incremental portionof the tissue section as it is being scanned. After the entire tissuesection is scanned, the system then presents the available images to theuser. Because, in most cases, not all of the video images can bedisplayed on the video display 33 at once, the computer 30 divides theimages into a number of non-overlapping visual fields. Each visual fieldconstitutes a mosaic of video frames representing a portion of thetissue section of interest and is displayed on the video display 33. Anexample of a visual field is illustrated on FIG. 3. The operator canreview all of the video fields until the entire region of interest hasbeen reviewed.

In accordance with the present invention, the target cells which are tobe either preserved or destroyed are identified using an automaticidentification mode and/or a manual identification mode. In theautomatic identification mode, the operator then sets the criteria bywhich the computer will automatically identify the target cells ofinterest to be ablated based on image characteristics. For example, thecriteria for automatic identification of target cells are based onobject color, hue, intensity, size, shape, texture, or any feature ofcombination of features definable by digitized image data.

The computer 30 then analyzes the video frames in accordance with theselection criteria and identifies cells falling within that selectioncriteria. The identified cells are contrasted from non-selected cells byany suitable manner such as by highlighting only those cells on videodisplay 33. At this stage, the operator may check the computerselections to see if the target cells of interest have been properlyidentified for ablation. If the operator wants to identify additionalcells to be ablated or deselect cells, the operator selects or deselectsthose cells by use, for example, of the light pen 34 on video display33, as described below in connection with manual identification.

In the manual identification mode, the operator uses the displayedimages to visually identify the target cells of interest using standardhistomorphological criteria known to persons having skill in the art. Afew examples of such target cells include identifiable cancer cells,identifiable muscle cells, or identifiable lymphocytes, etc., all ofwhich have well known appearances to those knowledgeable in the art.Features of visual appearance which may be considered by the operator ineither the manual or automatic identification mode are, for example,nuclear size, shape (e.g. roundness), texture and hue. The operator canalso choose cells based on a combination of features, such as theirvisual appearance and their location in the tissue under study.

In the manual identification mode, the operator visually selects thetarget cells of interest by “coloring in”, outlining or otherwiseidentifying those cells on the video monitor 33 using the light pen 34,illustrated in FIG. 3, or by using a mouse pointer, cursor system ortouch sensitive system (not shown). For example, the operator may draw acircle to contain the single gland of cancer (37) contained in FIG. 3,and upon completing this circle, the software will automatically changethe color of this circular region to indicate to the operator that ithas been selected. The operator will be able then to erase or alterthese selected regions as many times as necessary until the operator issatisfied with the selected regions.

When the operator is satisfied with the target cell selections in eachof the images in the first visual field (from either the automatic ormanual identification modes), the system immediately presents the secondvisual field to be viewed. When selections have been made for each imagein each visual field, the operator can review the images in any or allvisual fields. When the operator is satisfied with target cellselections from all of the images, the operator can then initiate theselective laser exposure process from the menu selections on display 33,utilizing terminal 36 (FIG. 4). The operator menu selections willcontain choices such as, for example, fluence level, beam width, energyexposure per unit selected area, magnification level and whether toexpose selected areas or expose non-selected areas. Before operating thesystem, these parameters will be set by the operator. After initialselection of laser treatment parameters, the operator will have theoption of saving these settings for subsequent use as default settings,to further streamline the laser treatment process.

After the selected target cells of interest have been identified asdiscussed above, the computer 30 analyzes the data and calculates thecourse of motion for the motorized stage. As a preferred embodiment, toincrease the speed of the photoablation process the software alsoanalyzes the size of the regions to be photoablated, and where wideareas are to be ablated, the widest appropriate beam diameter isautomatically applied based on the width of the area to be photoablated.The user then initiates a software-driven process whereby all of theselected cells are exposed to the UV laser automatically and unselectedcells are left unexposed. Verification of accurate lasing can beaccomplished in some cases by visible blanching of the tissue stainingdye in areas exposed to the laser, with preservation of staining innon-lased areas. Laser fixation points made of UV sensitive ink can beplaced on the slide as well, and can be used to verify accuracy.Appropriate shielding should be employed by the user to avoid exposureto the UV laser beam during use.

NIH-Image software, which is readily available, can be modified toprovide a convenient, user-friendly selection and ablation protocol asdescribed above and in accordance with the algorithm set forth in FIG.5.

The operator may stop photoablation after completion of this stage orproceed to higher magnification to obtain greater sample purity, ifdesired and/or necessary. Based on visual estimation, tumors with tissuepatterns consisting of concentrated clusters of cancer cells with awidth of 100 um or more, surrounded by 50-500 micron wide acellularareas or noncancerous cells can be effectively concentrated using alower power objective alone such as, for example, a 10× objective. Oneexample of such a tissue is a lymph node containing focal cancermetastases. The total time required for photoablation up to this stagedepends on the size of the tissue section examined, and the complexityof the selected regions. In the simplest case, for example a 1 cmdiameter lymph node containing four 1 mm diameter metastatic foci, theprocess would take from 2-5 minutes. Selective photodissection of abreast or prostate needle biopsy containing focal carcinoma would take asimilar amount of time.

If the operator wishes to further photodissect the areas left unexposed,the system is switched to a higher power objective lens such as, forexample, a 20×, 32× or 100× lens, and the slide is re-scanned. Regionswhich have been exposed to the laser can be identified by theirphotobleached appearance, giving the operator visual confirmation of theeffectiveness of photoablation and stage positioning. After scanning atthe higher magnification, the system can again provide a tiledrepresentation of these visual fields on the video display 33. Theoperator again chooses a mode of target selection, which will consist ofeither sequential viewing of each visual field and manually drawing theregions to be ablated, as described above, or by the use of theautomatic identification mode. The operator then reviews the results ofthe automated selection and, if satisfied with those selections, beginsphotoablation of the regions selected.

If more convenient, the operator may identify nuclei to be preserved andinstruct the system to destroy all other non-selected nuclei.

In one embodiment, when the operator desires to rescan the tissuesection using a higher magnification, the operator can choose to scanonly those portions of the tissue section that were not treated with thelaser at the lower magnification. In this embodiment, the computer 30recalls the coordinates of the regions photoablated in prior step andscans only regions of the tissue section that were not photoablatedpreviously. In tissue section regions located on the edge ofphotoablated areas, the visual fields presented to the operator on videodisplay 33 include a wide margin of previously photoablated tissue (suchas, for example, a 50 μm margin) to avoid skipping areas.

After a purified tissue section is obtained using the apparatus andprocess described above, the tissue material can then be placed in anappropriate solution for molecular analysis. For example, the treatedtissue section is removed from the slide upon which it is mounted usingsuitable means, such as a mechanical removal using a sterile scalpel, orby simple irrigation with appropriate buffer solution. The removedtissue is then placed in a suitable container such as a sterilemicrocentrifuge tube for molecular analysis.

The present invention enables accurate, efficient separation ofcontaminating cells from target cells of interest creating purified DNA,RNA or protein from tissue samples for molecular genetic analysis suchas PCR.

The laser purification system of the present invention can be used withBrightfield microscopy. Brightfield microscopy employs a broad-spectrumor white light source for illumination of appropriately stained tissuesections. The light source is typically placed opposite the objectivelens employed, with the tissue section to be examined placed between themicroscope objective and the light source. Brightfield microscopy can beused with either the automatic or manual identification mode, i.e. itwill allow the operator to visually select areas of interest in asemi-automated, interactive process based on image characteristics, asdiscussed above, or will permit the computer to automatically selecttarget cells of interest, as described above.

One of many possible automated selection methods using Brightfieldmicroscopy would involve the use of immunoperoxidase-stained tissuesections. Using immunoperoxidase staining, cells containing epitopesrecognized by a specific antibody are typically stained brown. If aspecific antibody is available for cells which the user wishes toselectively preserve or ablate within a tissue section, the entireselection process can be automated. For example, if the user wished toselectively study prostate epithelial cells within a tissue section,then the user could stain the tissue with a PSA-specific antibody usingthe immunoperoxidase technique, and stain all prostate epithelial cellsbrown (including both benign and malignant prostate cancer cells). Theuser then selects the scanning function, and after the tissue sectionhas been scanned, then selects the “automated” submenu. From theautomated submenu, the user would then choose criteria for objectselection, as described above. In this case, the user can select “brown”and “edge detection” which would trigger the software to find the outeredges of any object colored brown and select this area. The user canreview the selections made by the software prior to initiating the laserexposure process, as described above.

Another example of the operation of the present invention will bedescribed in connection with using fluorescence microscopy. Influorescence microscopy, cells are stained with a staining media whichwill fluoresce when illuminated by incident light, usually providing abright signal on a dark background. The incident light used is anultraviolet light source which excites fluorescent species within thestaining medium. Specific wavelengths of light produced by the objectswithin the tissue can be examined by selection of filters which allowlight of only defined spectra to pass.

Appropriate staining media will include, for example, propidium iodidewhich selectively stains DNA. Other media such as Fluoresceinisothiocyanate (FITC), Rhodamine, or Texas Red labelled antibodies ornewly developed more stable and intense fluorophore labels such as Cy3or Cy5 (Amersham, Inc.) labelled antibodies (or other probes) may beused with this method.

An immunofluorescence based function will allow nearly fully automatedphotoablation, with the user simply monitoring the initialidentification of target areas to be treated. The operator then directsthe system to either photoablate the area containing fluorescent signal,or photoablate areas not containing fluorescent signal. The operatordefines a fluorescence brightness threshold to determine the areas to betreated. Options for selection of target cells of interest usingfluorescence microscopy will be identical to those for brightfieldmicroscopy, including manual and automated identification methods. Theautomatic identification mode will operate similar to those used forimmunoperoxidase staining described above, and will allow the user toreview automated selections prior to selective laser treatment.

For example, the tissue section of interest is mounted on tissue support14 and covered with the quartz coverslip 15 as described above. Theoperator selects filters appropriate for the fluorophore in use. Theoperator then views the tissue section with a low power objective suchas, for example, 10× magnification, and prompts the system to scan thesection and select targets based on signal intensity. The operator thenreviews the selected targets field by field, and may decide to changethe intensity threshold for targeting if too many or too few targetshave been selected. Manual deselection or modification of targets inindividual visual fields also is possible as described above. If targetselection is not as precise as desired, the user may switch to a higherpower objective and repeat the process. When appropriate intensitycriteria for automatic target selection have been defined, based onreview of some or all of the selections by the operator, the operatortriggers the system to photoablate the selected regions. This processshould take from 2-10 minutes for most sections using a 10× objective,longer if high power objectives are used.

After target cell selection, the selected regions will be indicated onthe viewing screen either by a line circumscribing the region, or by achange in color or hue of the entire selected region, or by somecombination of line and color or hue that can be chosen by the user. Theuser will accept, reject, or modify these selections. Processing speedwill depend on the magnification used and the degree of selectivitydesired.

Different combinations of coverslips and mounting media may be used. Inone embodiment, a mildly UV absorbing mounting medium may be used toreduce laser scatter while allowing sufficient laser energy to reach thetarget. Tissue to be treated can be unfixed or fixed using any type offixative known to persons skilled in the art such as, for example,formalin, alcohol or acetone.

If studying tumor cells' DNA only, as is often done, the operator mayselect or deselect targets based on whether or not the cells arecancerous by visual determination, as described above. Since only targetnuclei need to be photoablated to produce selective DNA degradation, thehistological patterns of growth and nuclear characteristics typical ofmany cancers should be recognizable with nuclear staining alone,staining with hematoxylin alone, or toluidine blue alone to increaseimage contrast and allow clear recognition of nuclear boundaries.Automated nuclear roundness determination using Brightfield microscopycould also be performed more easily in samples so prepared.

In another embodiment of the present invention, a beam expander is notused and the device can be configured to function with a fixed beamwidth, such as 10 um, or any other convenient beam width for the desiredtissue or cells. Fixing at a diameter of 10 um should not interfere withcell-by-cell photodissection, since most mammalian cells are larger than10 um in diameter, however operation at a fixed beam diameter will slowthe photodissection process.

In still another embodiment, the present invention further comprises animage capture method and apparatus which enables the operator to selectspecific boundaries of the tissue section 12 to be scanned and treated,thereby significantly reducing time required to complete the overalllaser purification process.

In accordance with the image capture process of the present invention,an appropriately stained tissue section 12 is covered with a UVtransparent quartz coverslip 15 with suitable mounting media 13, asdescribed above. The assembled slide, tissue section and coverslip isplaced under the histologic microscope with attached high resolutioncamera 20. The operator selects the objective to be used whichpreferably is a low power magnification such as 2×, 4× or 10×. In oneembodiment, the operator then uses a toggle switch 11 to move themotorized stage in the x,y plane such that the tissue section appears onthe video monitor 33. The user then focuses the image on the videomonitor 33 using the microscope focusing knob. In a preferredembodiment, the image is automatically kept in focus during theprocedure through standard autofocus mechanisms known to those skilledin the art.

Viewing each visual field on the video display 33, the operator thenidentifies or marks specific boundaries of the tissue section to bescanned and treated. This may be performed manually by drawing a lineusing the mouse, light pen, touch screen, or cursor based system on thevideo monitor 33.

In another embodiment, the boundary identification can also beaccomplished automatically by using the mouse, light pen, touch screen,or cursor based system to select an automatic scan function on thecomputer screen. Activating the automatic scan function causes thesoftware to initiate movement of the stage in one direction. Using imageanalysis software, when the edge of the tissue section is reached, it isdetected and the coordinates of the edge are stored in computer memory.The software then causes the stage to be moved and to identify and mapthe entire outer perimeter of the section using stage motion algorithmsknown in the art. The entire outer edge of the section is mapped in thisfashion. Whether the edge is identified manually or automatically, theoperator will have the option of displaying the shape of the outerperimeter of the section on the video screen 33 to help confirm that theentire section has been scanned. The software also calculates the numberof captured images and corresponding number of visual fields needed toview the entire tissue section, and provides this information to theuser on-screen.

Along with providing the user with the number of and visual fieldsneeded to treat the selected portion of the tissue section or the entiretissue section, a menu of image capture options are provided on thevideo screen. The first option includes scanning and capturing images ofthe entire tissue section. These images are contiguous and mapped in thex,y plane by the software. The second option includes scanning andcapturing images from only a portion of the tissue section selected bythe operator. The third option includes scanning an initial portion ofthe section if adequate computer memory is not available. Targetselection and laser treatment of this initial area must then becompleted before the computer automatically reenters the image capturesequence for the next region. This is repeated until the entire sectionis treated.

In one embodiment, if the operator wishes to treat only specifiedportions of the section, the user chooses this option on the menu andselects an objective having a low magnification such as, for example, 2×or 4×. The software then triggers the motorized stage to quickly capturecontiguous low resolution images of the entire section. The tiled imagesrepresenting the entire tissue section are presented in one or morevisual fields on the video display 33, depending on the size of thetissue section. The operator then uses a mouse, light pen, touch screenor cursor system to select the tiles to be treated. After this selectionis made, the software prompts the user to capture images from theselected tiles at resolution adequate for target selection, which willbe determined by the user's preferences and needs. The target selectionand laser treatment are then carried out in accordance with the presentinvention, as described above.

Using the image capture process of the present invention allows forgreater control over scanning and treatment functions which can greatlyexpedite the laser purification process of the present invention.

In still another embodiment of the present invention, a manual methodand apparatus for purifying a tissue section by ablating the DNA, RNA orprotein of unwanted cells is disclosed which is more economical (butmore time consuming). In accordance with this embodiment, the UV lasercan be mounted on a standard microscope with motorized or purely manualmechanical microcontrol of beam direction. The tissue section, which cancomprise living and/or non-living cells, are supported by tissue support14 and are covered by quartz coverslip 15. Under direct vision (throughthe microscope) while wearing UV protective goggles, the user appliesthe laser beam to the areas desired. Areas lased are identified as areaswhere the dye used to stain the tissue has blanched. To prepare thelased tissue for analysis, the quartz coverslip is removed manually, andthe portion of tissue to be analyzed is physically removed from theslide using a sterile scalpel or scissors and placed in an appropriatereaction tube. The tissue is then placed in a buffer appropriate to theanalysis desired.

An automated calibration system for the motorized stage can be used tomeasure the precision of the stage motion system to allow for correctionof potential stage motion inaccuracies. In one such method, the surfaceof a test slide can be marked with the laser at high power, with adiffraction limited 5 micron diameter spot at a position withcoordinates x1, y1. The stage will then be moved repeatedly away fromx1, y1 to a fixed position x2, y2 and back to x1, y1. The position ofthe optical axis in relation x1, y1 will be measured by the imageanalysis system after each excursion using a cross hair in the viewingpath as the reference point. This process may be repeated as many timesas necessary to acquire a large data set for statistical analysis.Displacement from x1, y1 to x2, y2 can be varied to test whetherprecision varies with displacement. Stage motion is considered to behighly reproducible if there is an average of less than a two microndisplacement of the optical axis after 10 excursions. If stage motion ishighly reproducible as defined above, scanning and user-directed imageprocessing of the entire tissue section (including multiple images)could be performed in a single step, followed by photoablation ofselected areas in a second step. If stage motion is not highlyreproducible as defined above, in order to avoid photoablation ofunselected regions due to imprecise positioning, photoablation can beperformed after regions to be ablated are selected from each image,minimizing the number of excursions from the optical axis between thetime of image capture and photoablation.

A beam width control test routine also may be included which directs thesystem to produce beams of varying diameters such as, for example, 1,10, 20, 40, and 100 um in diameter in rows on a glass slide. Fluencelevels adequate to etch glass are used initially. Spot sizes aremeasured and analyzed by image analysis for mean values, roundness, andcoefficient of variation. To be certain that fluence level does notaffect beam width accuracy, a similar experiment may be performed at lowfluence (0.01-0.1 Joules/cm2) on a glass slide coated with commerciallyavailable fluorescence coated glass beads. Spot sizes can be measuredand statistically analyzed with fluorescence imaging.

The following examples further illustrate, but not limit the operationand application of the present invention.

EXAMPLE 1 Scanning and Separation of Tissue Containing IntimatelyAssociated but Genetically and Histologically Distinct Cell Populations

Surgically removed rejected allograft kidneys are infiltrated by largenumbers of lymphocytes from the recipient's immune system, so theycontain large numbers of cells from two different people in intimateassociation throughout the tissue.

To discriminate these two cell populations using the laser separationmethod of this invention, six micron sections of FFPE rejected kidneysamples histochemically stained with a combination of monoclonal mouseanti-human leucocyte common antigen (DAKO-LCA) and monoclonal mouseanti-human monocyte/macrophage CD68 (DAKO-CD68) using conditionsrecommended by the manufacturer. This combination of antibodies detectsepitopes in all intact or partially intact lymphocytes contained withinthe section. Detection is performed with goat anti-mouse secondaryantibodies linked to Cy3 (Oncor Image, Inc.), a very stable dye with anexcitation and emission spectrum similar to rhodamine. No backgroundstaining need be performed, which maximizes the signal to noise ratio inthe imaged section. Using this method of staining, it was confirmed thatlymphocytes within those rejected kidney sections produce an intense redfluorescent signal on immunostaining by this method. Using the laserseparation system configured to detect Cy3, all fluorescent signals inthe section can be identified. To effectuate selective ablation of theinvading lymphocytes within the sections, the system may be configuredto expose the signal producing cell and a 10 um rim around each signal.DNA from one antibody-stained but nonlaser treated section may beincluded as a control at each locus studied.

DNA is isolated separately from single 6 um hematoxylin and eosinstained sections from rejected transplant kidneys using the methoddescribed below in example 3. Amplification of the 115-128 base pair(“bp”) microsatellite polymorphism D18S61 is then performed using DNApurified from a section of a rejected transplant kidney. Products areobtained by amplifying DNA from two non-laser dissected rejected kidneysections, and both show 4 separate alleles, consistent with the presenceof DNA from two separate individuals. This confirms that two separategenetically distinct cell populations are present in this tissue sample.Amplification of DNA isolated through photodisection of tissue sectionsof rejected kidney (as described above) will reveal only 2 alleles,demonstrating effective separation of the two distinct, intimatelyassociated cell populations.

EXAMPLE 2 Separation of Tumor Cell in Prostate and Ovarian Biopsies

Histologic sections from frozen prostate cancer biopsies are imaged withlight microscopy and noncancerous cells are photoablated using theBrightfield analysis protocol described above. In addition, DNA may beisolated from adjacent non-photodissected sections and from noncanceroustissue from the same patients for comparison. In prostate cancersamples, microsatellite polymorphisms on chromosome 8p22 (currentlythought to be the most frequently deleted chromosomal segment inprostate cancer), including D8S261, D8S549, D8S602, and D8S264, forexample, as well as LPL on 8p21 may be studied using the method of thisinvention. In another mode of identification, fluorescent labelledsecondary antibodies (such as Cy3 listed above) may be used to detectcells stained with this antibody. The laser ablation system can beconfigured to ablate cells that do not exhibit these signals.Selectively purified prostate cancer cell RNA and DNA or protein fromthese sections is then obtained and analyzed.

Ovarian cancer samples also may be analyzed. Histologic sections frompapillary serous ovarian carcinomas may be analyzed and photodissectedusing brightfield microscopy, using the protocol described above. Thesesamples are analyzed for loss of heterozygosity using microsatelliteloci on chromosome 17q, which has shown loss of heterozygosity in 70% ofprimary tumors studied.

EXAMPLE 3 Purification of DNA from Tissue Sections

Sections are transferred to a sterile 1.5 ml eppendorf tube using asterile blade and deparaffinized with 400 ul xylene for 15 minutes withgentle shaking. The tubes are centrifuged at 10,000×g for 2 minutes andthe xylene removed with a sterile pipette. The material is washed threetime in 400 ul 100% ethanol. Samples are briefly dried in a vacuumcentrifuge until no remaining ethanol is seen. The samples are incubatedovernight in 50 ul of a digestion buffer containing 100 mM Tris-HCl, 4mM EDTA, and 0.5 mg/ml proteinase K, then boiled for 8 minutes toinactivate the enzyme and pulse centrifuged. One microliter of theproduct is used for the PCR.

PCR reactions and gel electrophoresis may be performed as described inthe art (Bova, Cancer Res. 53:3869, 1993). The forward primer isend-labelled for each microsatellite locus to be studied by combining5.0 ul primer (20 uM), 1.0 ul T4 polynucleotide kinase (10U/ul) (NewEngland Biolabs), 2.0 ul 10× kinase buffer (0.7M Tris HCl 100 mM MgCl₂,50 mM dithiothreitol pH 7.6), 7.0 ul sterile deionized water, and 5.0 ul[gamma⁻³²P] dATP and incubating at 37° C. for one hour. Afterincubation, the T4 kinase is denatured by heating to 68° C. for 20minutes.

Primer mix is prepared by adding 75 ul sterile deionized water and 5 ulof the reverse primer to the end-labelled forward primer. PCR isperformed by combining 1 ul DNA template, 1 ul primer mix, 0.05 ul TaqDNA polymerase (5U/ul) (Boehringer-Mannheim), 1.2 ul 10×PCR buffer (100mM Tris-HCl, 15 mM MgCl₂, 500 mM KCl, pH 8.3) (Boehringer-Mannheim), 1.2ul dNTP mix (equal volumes of dATP, dCTP, dGTP, and dTTP each at 10 mM),and 7.6 ul sterile water per reaction. The mix is prepared on ice, mixedwell, and covered with one drop mineral oil. The PCR tubes are preheatedto 94° C. prior to placing the reaction tubes or plates onto thepre-heated (94° C.) thermocycler (Hybaid Omnigene). After 2 minutes at94° C., 28 cycles of denaturation at 94° C.×60 seconds, annealing at 60°C. (D8S261, D2S123) or 55° C. (D18S61, D17S798)×30 seconds, andextension at 72° C.×30 seconds, with 2 seconds extension/cycle areperformed. The products are mixed with 12 ul of stop buffer containing95% formamide, 0.05% xylene cyanol, 0.05% Bromphenol blue, and 20 mMEDTA. Samples are heated to 94° C. for 3 minutes, then placed on ice,and loaded by 1 ul aliquots onto 6% acrylamide sequencing gelscontaining 8.0 M Urea. Gels are run for 1.5-3 hours depending on productsize. The gels are vacuum dried and exposed to Kodak Biomax film at roomtemperature or −70° C. depending on intensity of the signal detected bygeiger counter.

Using the apparatus and method of the present invention, the user canselect specific regions of the tissue section to be preserved. Thedevice will automatically ablate nonselected portions of the section,while selected cells' DNA or RNA will remain intact for subsequentanalysis by PCR or other genetic methods. By providing convenient laserpurification of tumor cells from tissue sections, the method andapparatus of the present invention will facilitate genetic research incancer, and will provide a tool for the accurate molecular diagnosis oftumors.

The present invention overcomes prior limitations by employing directvision video analysis of tissue sections integrated with a UV laser andimproved tissue handling techniques whereby the user can efficientlyobtain samples of tumor DNA or RNA of high purity, while at the sametime obtaining important histological information about the tissue. Byspecially configuring a standard microscope and stage motion system, acomputer, a compact, economical laser, and specialized software, thissystem will enhance pathologic practice and speed cancer research.Indeed, the present invention permits the examination, selection anddestruction of the DNA, RNA or protein of individual cells withheretofore unobtainable accuracy, simplicity and efficiency.

Since many modifications, variations and changes in detail may be madeto the described embodiments, it is intended that all matter in theforegoing description and shown in the accompanying drawings beinterpreted as illustrative and not in a limiting sense.

I claim:
 1. A method for purifying a heterogenous cell population in atissue section, said method comprising: (a) supporting a tissue sectionhaving a heterogenous population of cells on a tissue support under amicroscope with an objective for viewing said tissue section; (b)scanning said tissue section with a video camera and producing a mosaicof video images representing the tissue section; (c) displaying saidmosaic of video images representing the tissue section; (d) analyzingeach frame of said mosaic of video images based upon target cellselection criteria and selecting target cells of interest based uponsaid target cell selection criteria; and (e) automatically andselectively ablating each selected target cell utilizing a radiantenergy beam.
 2. The method of claim 1 further comprising the step ofautomatically analyzing each frame of said mosaic of video images basedupon target cell selection criteria and automatically selecting targetcells of interest based upon said target cell selection criteria.
 3. Themethod of claim 1 further comprising the step of visually analyzing eachframe of said mosaic of video images based upon target cell selectioncriteria and manually selecting target cells of interest based upon saidtarget cell selection criteria.
 4. The method of claim 2 furthercomprising the steps of manually selecting additional target cells ofinterest or manually deselecting target cells automatically selectedbased on said target cell selection criteria.
 5. The method of claim 1further comprising the steps of: (a) changing the magnification of saidmicroscope to a higher magnification; and (b) repeating steps (a)through (e) in claim
 10. 6. The method of claim 1 wherein the step ofautomatically and selectively ablating each selected target cellproduces a tissue section having relatively intact DNA, RNA or proteinfrom only the non-selected cells.
 7. A method for laser-treatingspecific target cells in a tissue section having a heterogenouspopulation of cells, comprising: (a) supporting said tissue section on atissue support under a microscope with an objective for viewing saidtissue section; (b) scanning said tissue section with a video camera toproduce a mosaic of video images representing said tissue section; (c)optionally displaying said mosaic of video images; (d) analyzing saidmosaic of video images based upon preselected target cell selectioncriteria; (e) selecting said specific target cells based uponpredetermined target cell selection criteria; and (f) laser-treatingsaid selected target cells.
 8. A method according to claim 7, whereinsaid analysis and selection are performed automatically by a computer.